Cell-penetrating peptides enhance peptide vaccine accumulation and persistence in lymph nodes to drive immunogenicity

Significance Current peptide antigen vaccine strategies have shown modest success in eliciting robust T cell priming. We defined mechanisms of action for a promising strategy based on covalently linking peptide antigens to cell-penetrating peptides (CPPs). We found that CPPs enhance CD8+ T cell priming through improved lymph trafficking/lymph node accumulation, higher uptake by antigen-presenting cells, increased peptide stability, and prolonged exposure of the antigen in the draining lymph node. Understanding the mechanisms by which CPPs enhance the immune response toward tumor antigens provides a foundation for future applications of peptide vaccines in combination immunotherapies.

Antigen-CPP click conjugates and fluorophore-labeled conjugates were purified in a similar manner post-reaction using MD RP-HPLC. Solvent was flowed at 4 mL/min over an Agilent Zorbax 300SB-C3 column (9.4 x 250 mm, 5 μm) using a linear gradient ramping from either 5 to 35% B or 15 to 45% B over 60 minutes, with fractions collected in 4 mL increments. Fractions were pooled and analyzed as above. All peptides and peptide conjugates were characterized via LC-MS to verify identity and purity.
Antigen-CPP conjugation. The indicated azide-containing long antigens were conjugated to each alkyne-CPP via Cu(I)catalyzed click chemistry. Approximately 1 μmol of each reagent was dissolved in 30:70 water: dimethyl sulfoxide (DMSO, v/v) that had been sparged with N2. Reactions were incubated for 1 hour on a nutating mixer, then quenched with the addition of 10 mL water with 0.1% TFA additive. Conjugates were then purified via reversed-phase HPLC as described above and isolated with >90% purity. Product identity and purity were verified via LC-MS. Fluorophore conjugation. Antigen or antigen-azide peptides (20 mg) were massed out into a 50 mL conical vial and dissolved in 45 mL water, then combined with equimolar BDP-TR-maleimide, FITC, or SulfoCy5-maleimide (50 mM stock in DMSO). Reactions were incubated for 30 minutes at room temperature on a nutating mixer, then filtered with a 0.22 μm syringe filter and purified by RP-HPLC as described above. Alternately, for the antigen-azide peptides, which were subsequently conjugated to various alkyne-CPPs and purified, the reaction mixture was lyophilized immediately without purification in order to minimize loss of material. Product identity and purity were verified via LC-MS.
LC-MS analysis. Unless otherwise stated, LC-MS analyses were performed on an Agilent 6520 ESI-QTOF mass spectrometer with an Agilent Zorbax column (300SB C3, 2.1 x 150 mm, 5 μm). Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in LC-MS grade acetonitrile. The LC-MS method was as follows: 1% B from 0 to 2 min, linear ramp from 1% B to 61% B from 2 to 11 min, 61% B to 99% B from 11 to 12 min and finally 3 min of post-time at 1% B for equilibration, flow rate: 0.8 mL/min. Some LC-MS analyses were performed on Agilent 6550 ESI-QTOF mass spectrometer with an Agilent Zorbax column (300SB-C3 2.1 x 150mm, 5 μm). Mobile phases were same as previous and the gradient was as follows: 1% B from 0 to 2min with a linear gradient from 1 to 95% B over 10 minutes, then 95% B for 1 minute with the MS on from 4 to 12 minutes and a flow rate of 0.5 mL/min. All Agilent system data were processed using the Agilent MassHunter software package. Y-axis in all chromatograms represents total ion current (TIC) unless noted.
LC-MS/MS analysis for the serum protein pulldown digest was performed using an EASY-nLC 1200 nano-LC system with an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (both Thermo Fisher Scientific) as described, using a PepMap RSLC C18 column (2 μm particle size, 15 cm × 50 μm ID; Thermo Fisher Scientific). Mobile phase A was 0.1% formic acid in ultrapure water (v/v) and mobile phase B was 80% acetonitrile, 0.1% formic acid in ultrapure water (v/v). The nano-LC method was as follows: 1% B ramping in a linear manner to 41% B in A over 120 minutes, flow rate: 300 nL/min. Data were processed using PEAKS proteomics software.
Serum stability LC-MS assay. Peptides were incubated at 5 µM in PBS with 5% mouse serum (Gibco) at 37 °C for 24 hours. At the indicated time points, a 5 μL aliquot of the reaction mixture was removed, transferred to a fresh 1.5 mL microcentrifuge tube, and lyophilized. These aliquots were then re-suspended in water with 0.1% TFA and analyzed via LC-MS using an Agilent 6550 ESI-QTOF and the method described above.
Proteolysis LC-MS Assay. Peptides were incubated at 5 µM in PBS with 0.5 ng proteinase K at 37 °C for 2 hours. At each time point, a 0.5 µL aliquot was removed, transferred to an LC-MS vial and flash frozen. They were then resuspended 50:50 water:acetonitrile with 0.1% trifluoroacetic acid and reaction analyzed on an Agilent 6550 iFunnel Q-TOF MS. Time points were taken at t = 0 min, 30 min, 60 min, and 120 min.

1.
A. J. Mijalis, et al., A fully automated flow-based approach for accelerated peptide synthesis. Nat. Chem. Biol. 13, 464-466 (2017). LGEAAGAAGFNVLLLIHEPSA AAGFNVLLL M47 GRGHLLGRLAAIVGKQVLLGRKVVVVR M48 SHCHWNDLAVIPAGVVHNWDFEPRKVS Figure S1. Representative cell trace violet proliferation histograms of pmel T cells after incubation with peptide loaded splenocytes. DCs were pulsed with peptide for 1 hour, before incubation with cell trace violet-labeled pmel T cells for 48 hours. Live cells were assessed for fluorescence and percentage division from the unstimulated control.

Figure S2. Comparing scrambled and original CPP sequences. A)
Standard sequences of pAntp and MPG (used throughout this manuscript) and sequences of their scrambled variants. A random scrambler tool was used to generate these sequences and they were inspected to ensure that biophysical patterns present in the original sequences were not present in the scrambled variant. B) Mice (n=5) were immunized with 25 µg CDN and 5 nmol of either the long antigen (gp100), the indicated antigen-CPP with a click linkage (c-n), or the antigen click-conjugated to a randomly scrambled version of the indicated CPP on day 0 and day 14. The percentage of IFN- + T cells was determined via ICS on Day 21. A one-way ANOVA was performed comparing each construct to gp100. *** p < 0.001, * p < 0.05, n.s. not significant. In addition, a t-test comparing the c-n and scrambled versions of each CPP indicated they were not statistically different (p > 0.05).   Figure S4. T cell response after stimulation with the optimal peptide epitope as determined by intracellular cytokine staining. Peripheral blood from 5 animals that had undergone prime and boost with either Adpgk-pAntp or Reps1-pAntp was stimulated with the matched cognate optimal epitope or a mismatched epitope, and then underwent intracellular staining to detect IFN-γ production by flow cytometry. Shown are mean ± standard deviation ***, p < 0.0002; ****, p < 0.0001 by 2way ANOVA followed by a Tukey post hoc test. , using standard pre-processing (Picard tools), alignment to the mm10 reference genome (bwa tool), and C57BL/6 tissue as the germline comparison. Displayed are the number of total variants (called by a consensus of mutect and strelka tools, and shared by both replicates), missense and InDel variants (identified by VEP annotations), and predicted strong MHC I binders (defined as ic50 binding affinity<1,000 and binding affinity percentile<0.5; using consensus predictions from NetMHCpan, NetMHC, and PickPocket tools)stratified by H2 alleles. Neoantigen candidates were prioritized from among predicted strong binders based on the variant allele frequency (VAF) in both DNA and RNA sequencing, gene expression (transcripts per million; TPM), and synthesizability of the neoantigen peptide. B) For screening of candidate neoantigens' immunogenicity, C57BL/6 mice were immunized subcutaneously with 100 µg of select neoantigen minimal epitopes (dissolved in DMSO) and 100 µg of Complete Freund's Adjuvant. Spleens were collected on day 10 and processed for IFNγ ELISpot (Mabtech, AB.). Representative responses are displayed (200,000 splenocytes per well, stimulated with cognate minimal epitopes for 36-48 hours), and compared to negative (SIINFEKL peptide) and positive (phytohemagglutinin) controls. C) Three confirmed neoantigens (all missense variants) were selected for further experimentationincluding one from GL261, one from CT-2A, and one present in both lines. For each, 21-mer synthetic long peptide (SLP) and 37-mer cell penetrating peptide (CPP) versions were synthesized (GenScript, Co.; with all SLP and CPP purities confirmed by HPLC to be ≥90%). The neoantigen's amino acid substitution is indicated by bold underline. The 21-mer SLP includes the minimal epitope sequence (red) and flanking native peptide sequence (black). The CPP version consists of the SLP with a C-terminal RQIKIWFQNRRMKWKK sequence (blue). Average VAF and TPM values are reported from two replicates. Figure S6. Exhaustion phenotyping on peripheral blood after a prime and boost in naïve mice. C57Bl/6 mice (n = 5 animals/group) were immunized with 5 nmol Adpgk or Adpgk-pAntp peptide combined with 25 µg cyclic-di-GMP on days 0 and 14, and then on day 21 peripheral blood was restimulated with optimal Adpgk peptide ex vivo, permeabilized for intracellular cytokine staining to identify antigen-specific IFN- + cells and stained for cell surface phenotypic markers. Representative flow cytometry plots, percentages, and quantification of PD-1/TIM3 expression. Statistical analyses were performed using a two-way ANOVA with Šídák's multiple comparisons test.    B.
Radiant efficiency Figure S12. Identification of serum proteins that bind antigen-CPPs. A) First wash, final wash, and elution native PAGE gels from experiment in figure 5D. Briefly, biotinylated CPPs were bound to streptavidin beads, then incubated in 50% mouse serum for 1 hour. The beads were then washed using PBS, analyzing each wash via native PAGE until no further serum proteins were observed in the eluate. Bound proteins were then eluted in mild acid and analyzed by native PAGE. B) Several prominent bands were excised from the elution gel in A, digested in trypsin, and analyzed via LC-MS/MS. PEAKS software was used to identify the proteins present in each band. Figure S13. CPP conjugation confers protection in serum relative to the antigen alone. Extended serum stability data from Figure 6A-C. Each construct was either used fresh or incubated overnight in 10% serum, then serially diluted and used to stimulate pmel T cells in an in vitro activation assay. Shown are plots of pmel activation, measured via CD69 upregulation, against the concentration of the fresh or serum-treated construct. The difference between the fresh and serum treated plots indicates the extent of degradation in serum. This difference can also be visualized by plotting the log(EC50) of the fresh and serum-treated samples for each construct.  Figure S14. CPP conjugation does not confer protection against proteolysis by isolated proteinase K. Peptides (gp100 and gp100-MPG) were incubated at 5 µM in PBS with 0.5 ng proteinase K for 120 minutes. At 0, 20, 40, 60, and 120 min 5 µL aliquots were removed and analyzed via LC-MS to determine the relative amount of intact antigen construct remaining. Figure S15. Mice were immunized with 25 µg CDN and 5 nmol of either EGP or EGP-pAntp on Days 0, 6, 10, or 13 (one group per time point). On day 14, 10 6 pmel-1 T cells were injected retro-orbitally. 24 hours later, axillary and inguinal lymph nodes were harvested and analyzed to determine CD69 upregulation. Data are shown for the axillary lymph nodes and the control line is derived from untreated spleens (inguinal lymph node data are given in Figure 6F).